Inactivation of the p53 tumor suppressor protein is considered essential for the development of most or all human cancers. Over 50% of cancers harbor TP53 mutations that destroy p53 function. In most cases, missense mutations in one TP53 allele are followed by loss-of-heterozygosity (LOH), so tumors express only a mutant, inactive form of p53. TP53 mutations and LOH have been linked, in many cases, with poor therapy response, increased tumor aggressiveness, and decreased long-term survival. Despite this, remarkably little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. Approximately 15% of human colorectal cancers (CRCs) are microsatellite unstable (MSI) due to somatic or inherited defects in DNA mismatch repair (MMR). Interestingly, TP53 mutations are less frequent in MSI (MMR deficient) CRCs than cancers where MMR is intact. This has suggested MMR defects do not give rise to TP53 mutations in MSI tumorigenesis, though experiments to directly address this question are lacking. Wt p53 is expressed at low levels due to MDM2, an E3-ligase that binds p53 and promotes its degradation. Small molecule MDM2 antagonists are being developed as therapeutic agents. Nutlin-3a (Nutlin) is an MDM2 antagonist that stabilizes/activates p53 by blocking MDM2-p53 binding. Nutlin causes robust growth arrest/apoptosis in p53 wt cells and human tumor xenografts. Importantly, only p53-null or p53-mutant cells can grow and form colonies in Nutlin. In this grant proposal, we have leveraged the powerful growth inhibitory activity of Nutlin to select p53-mutated cells and examine how TP53 mutations arise and how LOH occurs. Preliminary data suggest MMR-deficient CRC cells acquire TP53 mutations at greater rate than repair-proficient counterparts. Further, these initiating TP53 mutations appear more prevalent in cells that express certain cancer stem cell (CSC) markers (CD44), but not CSC marker-negative cells. Finally, TP53 LOH in MSI cells occurs through a copy-neutral mechanism. Based on these findings we hypothesize 1) MMR deficiency gives rise to initiating point mutations in p53, 2) TP53 LOH in MSI cells occurs through a copy-neutral mechanism, and 3) CD44(+) are either more prone to acquire TP53 mutations, or p53 mutations convert CD44low cells into CD44hi and thus may increase cancer stemness.